Proteolytic enzyme activity of organic and mineral soil core samples collected near Toolik Lake field station, Alaska, July 2001

The original focus of this study was an analysis of proteolytic enzyme activity of Alaskan arctic tundra soils, however initial results raised questions regarding the method (Watanabe and Hayano, 1995). Thus, the goals of the study changed to 1) an investigation of the method, and 2) a comparison of enzyme activities of two different soil layers from the arctic tundra. Methodological examination included the impact of toluene, used to prevent immobilization of the product, and blank correction of enzyme activity, and a search for a true 6-h linear rate of activity during a 48-hour incubation. We measured native and potential, using casein as an artificial substrate, activities as net amino acid production in mineral and organic soil layer samples. Varying toluene concentration had no clear effect on activity; omitting toluene resulted in zero native activity and reduced potential for the organic samples, but not for the mineral. Comparison of activities with and without blank correction indicated, particularly for potential activity of samples with low native rates, that correction was required for accuracy. Native and potential activity of the organic samples, and native of the mineral were linear for the first 6 h of incubation; linearity was observed during the 6 to 24 h incubation for potential activity of the mineral. Soil layer activity data indicated that native activity was higher in organic soils as compared with mineral. The organic layer potential activity was ten-fold greater than the native, suggesting substrate limitation; potential and native activities did not differ in the mineral layer, indicating substrate sufficiency. Casein addition changed the kinetic pattern for both layers from hyperbolic to sigmoidal for the mineral and linear for the organic, implying different enzyme pools or behavioral changes of existing pools. Native activity based on total soluble protein was higher for the mineral samples relative to the organic, reiterating substrate capacity differences and variations in enzyme/substrate interactions.本研究最初的核心目标为分析阿拉斯加北极苔原土壤的蛋白酶（proteolytic enzyme）活性，但初步结果引发了对实验方法（Watanabe与Hayano，1995）的质疑。因此，本研究的研究目标调整为：1）对该实验方法开展验证性考察；2）对比北极苔原两种不同土壤层的酶活性差异。 方法学验证环节涵盖了甲苯（toluene，用于防止产物固定化）的影响、酶活性的空白校正，以及探究48小时孵育过程中真实的6小时线性活性速率。本研究以酪蛋白（casein）作为人工底物，测定矿质土壤层与有机土壤层样品的本底活性与潜在活性，以净氨基酸生成量作为活性评价指标。 不同浓度的甲苯对酶活性未产生明确影响；但省略甲苯时，有机样品的本底活性归零，且其潜在活性受到抑制，而矿质样品未出现该现象。对比经空白校正与未经空白校正的活性数据后发现，尤其针对本底活性较低的样品的潜在活性，空白校正对保证结果准确性不可或缺。 有机样品的本底与潜在活性、矿质样品的本底活性，在孵育的前6小时内均呈线性变化；矿质样品的潜在活性则在6至24小时的孵育期间呈现线性特征。不同土壤层的活性数据显示，有机土壤的本底活性高于矿质土壤。有机层的潜在活性较本底活性高出一个数量级，提示存在底物限制；而矿质层的潜在活性与本底活性无显著差异，表明底物供应充足。 添加酪蛋白后，两层土壤的动力学模式均发生改变：矿质层从双曲线型转变为S型，有机层则变为线性，这暗示二者存在不同的酶库，或现有酶库的行为模式存在差异。以总可溶性蛋白计算的本底活性显示，矿质样品的活性高于有机样品，进一步印证了底物容量差异以及酶-底物相互作用的异质性。